Enhanced expression and stability regions

ABSTRACT

Expression-enhancing nucleotide sequences for expression in eukaryotic systems are provided that allow for enhanced and stable expression of recombinant proteins in eukaryotic cells. Enhanced expression and stability regions (EESYRs) are provided for expression of a gene of interest in a eukaryotic cell. Chromosomal loci, sequences, and vectors are provided for enhanced and stable expression of genes in eukaryotic cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/752,647, filed on Jan. 23, 2013, now allowed, which is a divisionalof U.S. application Ser. No. 12/793,898, filed on 4 Jun. 2010, now U.S.Pat. No. 8,389,239, which is a continuation of U.S. non-provisionalapplication Ser. No. 12/132,846, now U.S. Pat. No. 7,771,997, filed 4Jun. 2008, which claims benefit under 35 USC 119(e) of U.S. ProvisionalApplication No. 60/933,213, filed 4 Jun. 2007, all of which are hereinspecifically incorporated by reference in their entirety.

SEQUENCE LISTING

This application hereby incorporates by reference the Sequence Listingsubmitted in Computer Readable Form as file 3051B_ST25.txt created onSep. 18, 2012 (79,397 bytes).

BACKGROUND

1. Field of the Invention

The invention provides for expressing recombinant proteins in eukaryoticcells. In particular, the invention includes methods and compositionsfor improved expression of proteins in eukaryotic cells by employingexpression-enhancing nucleotide sequences. The invention includesenhanced expression and stability region (EESYR) sequences thatfacilitate enhanced and stable expression of recombinant proteins ineukaryotic cells, and methods of using such sequences.

2. Description of Related Art

The development of expression systems is an important goal for providinga reliable and efficient source of a given protein for research andtherapeutic use. Recombinant protein expression in mammalian cells isoften preferred for manufacturing therapeutic proteins due to, forexample, the ability of mammalian expression systems to appropriatelypost-translationally modify recombinant proteins.

Several vectors are available for expression in mammalian hosts, eachcontaining various combinations of cis- and, in some cases,trans-regulatory elements to achieve high levels of recombinant proteinwith short incubation times. Despite the availability of numerous suchvectors, the expression of a recombinant protein achieved in mammaliansystems is often unacceptably low or otherwise unsatisfactory. Moreover,developing a cell line that reliably expresses sufficiently high levelsof a desired protein often requires time consuming cloning andamplification steps. Accordingly, there is a need in the art forimproved mammalian expression systems.

BRIEF SUMMARY

In one aspect, an isolated nucleotide sequence comprising anexpression-enhancing sequence selected from a sequence of SEQ ID NO:1-6,or an expression-enhancing fragment thereof, is provided.

In one embodiment, the expression-enhancing sequence comprises anexpression-enhancing sequence of SEQ ID NO:5 located at a positionwithin SEQ ID NO:5 selected from nucleotides spanning positions numbered10-13,515; 20-12,020; 1,020-11,020; 2,020-10,020; 3,020-9,020;4,020-8,020; 5,020-7,020; 6,020-6,920; 6,120-6,820; 6,220-6,720;6,320-6,620; 6,420-6,520; 6,460-6,500; 6,470-6,490; and 6,475-6,485. Inanother embodiment, an expression-enhancing sequence is provided that isselected from the group consisting of nucleotides 5,000-7,400 of SEQ IDNO:5; 5,000-6,500 of SEQ ID NO:5; 6,400-7,400 of SEQ ID NO:5; andnucleotides 6,400-6,500 of SEQ ID NO:5.

In another embodiment, the recombination recognition site is positionedas described above, providing that the expression-enhancing sequencecomprises a sequence that is at least 90% identical, more preferably atleast 95% identical, most preferably at least 99% identical, to theexpression-enhancing sequence of SEQ ID NO:5 or an expression-enhancingfragment thereof.

In one embodiment, the expression-enhancing sequence further comprisesat least one recombinase recognition site comprising a sequenceindependently selected from a loxp site, a lox 511 site, a lox 2272site, and a frt site. In one embodiment, the recombinase recognitionsite is within the expression-enhancing sequence. In another embodiment,the recombinase recognition site is immediately adjacent in the 5′direction to the terminal nucleotide of the 5′ end or immediatelyadjacent in the 3′ direction to the terminal nucleotide of the 3′ end ofthe expression-enhancing sequence.

In one embodiment, at least two recombinase recognition sites arepresent within the expression-enhancing sequence. In a specificembodiment, two recombinase recognition sites of opposite orientationare present within the expression-enhancing sequence. In anotherembodiment, three recombinase recognition sites are present within theexpression-enhancing sequence.

In one aspect, an isolated nucleotide sequence is provided thatcomprises an expression-enhancing sequence that is at least 80%identical, preferably at least 90% identical, more preferably at least95% identical, most preferably at least 99% identical to anexpression-enhancing sequence of SEQ ID NO:1-6 or anexpression-enhancing fragment thereof. In one embodiment, theexpression-enhancing sequence displays the recited identity to asequence of SEQ ID NO:5 as described above.

In one aspect, an isolated eukaryotic cell is provided that comprises anexpression-enhancing sequence selected from SEQ ID NO:1-6 or anexpression-enhancing fragment thereof. In one embodiment, theexpression-enhancing sequence comprises an expression enhancing sequenceof SEQ ID NO:5 as described above.

In one embodiment, the eukaryotic cell is a mouse, rat, hamster, orhuman cell. In a specific embodiment, the eukaryotic cell is a CHO cell.

In one embodiment, the eukaryotic cell further comprises at least onerecombinase recognition sequence within the expression-enhancingsequence. In a specific embodiment, the at least one recombinaserecognition sequence is independently selected from a loxp site, a lox511 site, a lox 2272 site, and a frt site. In one embodiment, therecombinase recognition site is immediately adjacent in the 5′ directionto the terminal nucleotide of the 5′ end or immediately adjacent in the3′ direction to the terminal nucleotide of the 3′ end of theexpression-enhancing sequence.

In one embodiment, at least two recombinase recognition sites arepresent within the expression-enhancing sequence. In a specificembodiment, two recombinase recognition sites are of oppositeorientation and are present within the expression-enhancing sequence. Inanother embodiment, three recombinase recognition sequences are present,and one of the three recombinase recognition sequences is in anorientation opposite to the two remaining recombinase recognitionsequences.

In one embodiment, the recombinase recognition site in theexpression-enhancing sequence of SEQ ID NO:5 is located at a positionwithin SEQ ID NO:5 selected from nucleotides spanning positions numbered10-13,515; 20-12,020; 1,020-11,020; 2,020-10,020; 3,020-9,020;4,020-8,020; 5,020-7,020; 6,020-6,920; 6,120-6,820; 6,220-6,720;6,320-6,620; 6,420-6,520; 6,460-6,500; 6,470-6,490; and 6,475-6,485. Inanother embodiment, the recombinase recognition site in a sequence thatis selected from the group consisting of nucleotides 5,000-7,400 of SEQID NO:5; 5,000-6,500 of SEQ ID NO:5; 6,400-7,400 of SEQ ID NO:5; andnucleotides 6,400-6,500 of SEQ ID NO:5. In a specific embodiment, therecombinase recognition site is located within nucleotides 6400-6500 ofSEQ ID NO:5. In another specific embodiment, the recombinase recognitionsite is inserted before, after, or within the “act” triplet ofnucleotides 6471 to 6473 of SEQ ID NO:5 in an expression-enhancingsequence of SEQ ID NO:5.

In another specific embodiment, the recombination recognition site ispositioned as described above, with the caveat that theexpression-enhancing sequence comprises a sequence that is at least 90%identical, more preferably at least 95% identical, most preferably atleast 99% identical, to nucleotides 5218 through 6048 of SEQ ID NO:5 oran expression-enhancing fragment thereof.

In a specific embodiment, the cell is a CHO cell and the recombinaserecognition site is inserted in the CHO cell genome at or within the“act” triplet of nucleotides 6,471 to 6,473 of SEQ ID NO:5.

In one embodiment, a first GOI is inserted within theexpression-enhancing sequence of SEQ ID NO:5 as described above, and thefirst GOI is optionally operably linked to a promoter, wherein thepromoter-linked GOI (or the GOD is flanked 5′ by a first recombinaserecognition site and 3′ by a second recombinase recognition site. Inanother embodiment, a second GOI is inserted 3′ of the secondrecombinase recognition site, and the second GOI is flanked 3′ by athird recombinase recognition site.

In a specific embodiment, the GOI is operably linked to a promotercapable of driving expression of the GOI, wherein the promoter comprisesa eukaryotic promoter that is regulatable by an activator or inhibitor.In another specific embodiment, the eukaryotic promoter is operablylinked to a prokaryotic operator, and the eukaryotic cell optionallyfurther comprises a prokaryotic repressor protein.

In another embodiment, one or more selectable markers are includedbetween the first and the second and/or the second and the thirdrecombinase recognition sites. In a specific embodiment, the firstand/or the second genes of interest and/or the one or more selectablemarkers are operably linked to a promoter, wherein the promoter may bethe same or different. In a specific embodiment, the promoter comprisesa eukaryotic promoter (such as, for example, a CMV promoter), optionallycontrolled by a prokaryotic operator (such as, for example, a tetoperator). In a specific embodiment, the cell further comprises a geneencoding a prokaryotic repressor (such as, for example, a tetrepressor).

In another embodiment, the cell further comprises a gene capable ofexpressing a recombinase. In a specific embodiment, the recombinase is aCre recombinase.

In one aspect, an isolated eukaryotic cell is provided that comprises anexpression-enhancing sequence that is at least 80%, more preferably atleast 90%, more preferably at least 95%, most preferably at least 99%identical to an expression-enhancing sequence of SEQ ID NO:1-6 or anexpression-enhancing fragment thereof. In a specific embodiment,expression-enhancing sequence is a sequence within SEQ ID NO:5 asdescribed above.

In one aspect, a eukaryotic host cell is provided, comprising anexpression-enhancing sequence selected from SEQ ID NO:1-6 or anexpression-enhancing fragment thereof, comprising a first recombinaserecognition site followed by a first eukaryotic promoter, a first markergene, a second eukaryotic promoter, a second marker gene, a secondrecombinase recognition site, a third eukaryotic promoter, a thirdmarker gene, and a third recombinase recognition site. In oneembodiment, the expression-enhancing sequence is within SEQ ID NO:5 asdescribed above.

In one embodiment, the first, second, and third recombinase recognitionsites are different. In a specific embodiment, the recombinaserecognition sites are selected from a loxp site, a lox 511 site, a lox2272 site, and a frt site.

In one embodiment, the first marker gene is a drug resistance gene. In aspecific embodiment, the drug resistance gene is a puromycin resistancegene. In another embodiment, the second and third marker genes encodetwo different fluorescent proteins. In one embodiment, the two differentfluorescent proteins are selected from Discosoma coral (DsRed), greenfluorescent protein (GFP), enhanced green fluorescent protein (eGFP),cyano fluorescent protein (CFP), enhanced cyano fluorescent protein(eCFP), and yellow fluorescent protein (YFP). In a specific embodiment,the two different fluorescent proteins are eCFP and DsRed.

In one embodiment, the first, second, and third promoters are the same.In another embodiment, the first, second, and third promoters aredifferent. In another embodiment, the first promoter is different fromthe second and third promoters, and the second and third promoters arethe same. In a specific embodiment, the first promoter is an SV40 latepromoter, and the second an third promoters are each a human CMVpromoter.

In one aspect, a eukaryotic host cell is provided, comprising anexpression-enhancing sequence selected from SEQ ID NO:1-6, or anexpression-enhancing fragment thereof, at least one recombinaserecognition site within the expression-enhancing sequence, and at leastone gene of interest (GOI) within the expression-enhancing sequence. Inone embodiment, the expression-enhancing sequence is a sequence withinSEQ ID NO:5, as described above.

In one embodiment, the cell comprises a first recombinase recognitionsite followed by a first promoter operably linked to a first GOI. Inanother embodiment, the first GOI is followed by a second recombinaserecognition site. In another embodiment, the second recombinaserecognition site is followed by a second promoter operably linked to asecond GOI. In another embodiment, the second GOI is followed by a thirdrecombinase recognition site. In another embodiment, at least one markeris operably linked to a third promoter and is located between the secondrecombinase recognition site and the second promoter. In one embodiment,the first recombinase recognition site is oriented in an oppositeorientation to the second and third recombinase recognition sites. Inone embodiment the first and second promoters are eukaryotic promotersoperably linked to a prokaryotic operator. In one embodiment, the firstand second promoters are CMV promoters operably linked to tet operatorsequences. In another embodiment, the cell further comprises a genecapable of expressing a prokaryotic repressor. In one embodiment, theprokaryotic repressor is a tet repressor. In one embodiment, the cellcomprises a gene capable of expressing a Cre recombinase.

In one embodiment, a first and a second marker gene are located betweenthe second recombinase recognition site and the second promoter, and anIRES is between the first and second marker genes. In anotherembodiment, the first codon (ATG) of the first marker gene isimmediately 5′ to the second recombinase recognition site, and thesecond codon of the first marker gene is immediately 3′ to the secondrecombinase recognition site. In another embodiment, the first markergene contains an intron and the second recombinase recognition site islocated within the intron such that the amino-terminal half of the firstmarker gene and the 5′ half of the intron are located 5′ of the secondrecombinase recognition site and the 3′ half of the intron andcarboxy-terminal half of the first marker gene are immediately 3′ to thesecond recombinase recognition site.

In one embodiment, the first, second, and third recombinase recognitionsites are different. In a specific embodiment, the recombinaserecognition sites are selected from a loxp site, a lox 511 site, a lox2272 site, and a frt site.

In one embodiment, the first marker gene is a drug resistance gene. In aspecific embodiment, the drug resistance gene is a hygromycin resistancegene. In another embodiment, the second marker gene encodes afluorescent protein. In one embodiment, the fluorescent protein isselected from DsRed, GFP, eGFP, CFP, eCFP, and YFP.

In one embodiment, the first, second, and third promoters are the same.In another embodiment, the first, second, and third promoters aredifferent. In another embodiment, the third promoter is different fromthe first and second promoters, and the first and second promoters arethe same. In a specific embodiment, the third promoter is an SV40 latepromoter, and the first and second promoters are each a human CMVpromoter.

In one embodiment, the first and second promoters are operably linked toa prokaryotic operator. In a specific embodiment, the operator is a tetoperator.

In one embodiment, the host cell line has an exogenously added geneencoding a recombinase integrated into its genome, operably linked to apromoter. In a specific embodiment, the recombinase is Cre recombinase.In another embodiment, the host cell has a gene encoding a regulatoryprotein integrated into its genome, operably linked to a promoter. In aspecific embodiment, the regulatory protein is a tet repressor protein.

In one embodiment, the first GOI and the second GOI encode a lightchain, or fragment thereof, of an antibody or a heavy chain, or fragmentthereof, of an antibody. In a specific embodiment, the first GOI encodesa light chain of an antibody and the second GOI encodes a heavy chain ofan antibody.

In one aspect, a method is provided for making a protein of interest,comprising: (a) providing a host cell that comprises anexpression-enhancing sequence selected from a sequence of SEQ ID NO:1-6;(b) introducing into the host cell, within the expression-enhancingsequence, a gene of interest (GOI) operably linked to a promoter; (c)maintaining the host cell of (a) under conditions that allow the GOI toexpress a protein of interest; and, (c) recovering the protein ofinterest.

In one embodiment, the cell is a CHO cell and the nucleotide sequence isan expression-enhancing sequence of SEQ ID NO:5 as described above.

In one embodiment, the GOI is introduced into the cell employing atargeting vector for homologous recombination, wherein the targetingvector comprises a 5′ homology arm homologous to a sequence present inat least one of SEQ ID NO:1-6, a GOI, and a 3′ homology arm homologousto a sequence present in at least one of SEQ ID NO:1-6. In anotherembodiment, the construct further comprises two, three, four, or five ormore genes of interest. In another embodiment, one or more of the genesof interest are operably linked to a promoter.

In another embodiment, the GOI is introduced employing an integrasetechnology, for example, integrase technology employing att sites suchas Invitrogen's Gateway™ and Multisite Gateway™ cloning systems whichemploy bacteriophage lambda att site recombination.

In another embodiment, the expression-enhancing sequence comprises oneor more recombinase recognition sites as described above, and the GOI isintroduced into the expression-enhancing sequence through the action ofa recombinase that recognizes the recombinase recognition site.

In one embodiment, the expression-enhancing sequence comprises tworecombinase recognition sites.

In one embodiment, the expression-enhancing sequence comprises a first,a second, and a third recombinase recognition site. In one embodiment,the first, second, and third recombinase recognition sites aredifferent. In another embodiment, the first, second, and thirdrecombinase recognition sites are not in the same orientation. In aspecific embodiment, the first site is 5′ to the second site, and thesecond site is 5′ to the third site. In another specific embodiment, thesecond and third sites are in opposite orientation with respect to thefirst site.

In another embodiment, a first and a second GOI are introduced into theexpression-enhancing sequence. In one embodiment, the first GOI isintroduced between the first and the second recombinase recognitionsites, and the second GOI is introduced between the second and the thirdrecombinase recognition sites. In a specific embodiment, the recombinaserecognition sites are independently selected from a loxp site, a lox 511site, a lox 2272 site, and a frt site.

In another embodiment, the first GOI flanked by recombinase recognitionsites on a first vector and the second GOI flanked by recombinaserecognition sites on a second vector are introduced into a cellcomprising an expression-enhancing sequence that comprises threerecombinase recognition sites in a single step.

In one embodiment, the GOI is operably linked to a eukaryotic promoter.In another embodiment, the eukaryotic promoter is operably linked to aprokaryotic operator. In a specific embodiment, the eukaryotic promoteris a CMV promoter and the prokaryotic operator is a tet operator.

In another embodiment, the cell comprises a gene capable of expressing aprokaryotic repressor. In a specific embodiment, the prokaryoticrepressor is a tet repressor.

In another embodiment, the cell comprises a gene capable of expressing arecombinase. In a specific embodiment, the recombinase is a Crerecombinase.

In one aspect, a eukaryotic cell is provided, wherein the eukaryoticcell comprises at least one expression-enhancing sequence of SEQ IDNO:1-6 and at least one exogenously added gene within theexpression-enhancing sequence. In one embodiment, the exogenously addedgene is operably linked to an exogenously added promoter.

In one embodiment, the expression-enhancing sequence is a sequence ofSEQ ID NO:5, and the at least one exogenously added gene integrated orinserted within the expression-enhancing sequence is a human gene. In aspecific embodiment, the eukaryotic cell is a CHO cell, the exogenouslyadded gene is a human gene, and the human gene is operably linked to anexogenously added eukaryotic promoter.

In one aspect, a targeting vector for homologous recombination isprovided, wherein the targeting vector comprises a 5′ homology arm, aGOI, and a 3′ homology arm, wherein each homology arm is homologous to asequence within one of SEQ ID NO:1-6. In one embodiment, the 5′ and 3′homology arms are homologous to sequences within SEQ ID NO:5. In oneembodiment, the targeting vector comprises two, three, four, or five ormore genes of interest. In one embodiment, the GOI is operably linked toa promoter. In another embodiment, each of the two, three, four, or fiveor more genes of interest are each operably linked to a promoter.

In one aspect, an expression vector is provided, comprising anexpression-enhancing nucleotide sequence selected from SEQ ID NO: 1-6 oran expression-enhancing fragment thereof. In one embodiment, theexpression-enhancing sequence is within SEQ ID NO:5 as described above.

In one embodiment, the vector further comprises a promoter. In aspecific embodiment, the promoter is a human CMV promoter.

In one embodiment, the vector further comprises a cloning site for anexpressible gene of interest (GOI). In one embodiment, the nucleotidesequence selected from SEQ ID NO: 1-6 or expression-enhancing fragmentthereof is located 3′ with respect to the coning site for theexpressible GOI. In another embodiment, the nucleotide sequence selectedfrom SEQ ID NO: 1-6 or expression-enhancing fragment thereof is located5′ with respect to the coning site for the expressible GOI.

In a specific embodiment, the vector comprises a nucleotide sequenceselected from SEQ ID NO:1-6 or an expression-enhancing fragment, a humanCMV promoter, a GOI, and a termination sequence, wherein the nucleotidesequence selected from SEQ ID NO: 1-6, or the expression-enhancingfragment thereof, is located 5′ with respect to the CMV promoter. In aspecific embodiment, the vector further comprises an intron selectedfrom a CMV-MIE intron and a rabbit β-globin intron.

In one aspect, an expression vector is provided, comprising anexpression-enhancing nucleotide sequence that is at least 80% identical,preferably at least 90% identical, more preferably at least 95%identical, most preferably at least 99% identical to a sequence selectedfrom SEQ ID NO:1-6 or an expression-enhancing fragment thereof.

In one aspect, a method is provided for making a protein of interest,comprising: (a) introducing into a host cell an expression vectorcomprising an expression-enhancing sequence selected from a sequencewithin SEQ ID NO:1-6 and a GOI that encodes for a protein of interest,wherein the GOI is operably linked to a promoter and operably linked tothe expression-enhancing sequence; (b) culturing the host cell of (a)under conditions that allow expression of the GOI; and (c) recoveringthe protein of interest.

In one embodiment, the enhanced expression and stability region sequenceis an expression-enhancing sequence of SEQ ID NO:1-6. In one embodiment,the expression-enhancing sequence is within SEQ ID NO:5 as describedabove.

In one embodiment, the recombinant protein is selected from the groupconsisting of a subunit of an immunoglobulin or fragment thereof and areceptor or ligand-binding fragment thereof. In a specific embodiment,the recombinant protein is selected from the group consisting of anantibody light chain or antigen-specific fragment thereof, and anantibody heavy chain or antigen-specific fragment thereof.

In any of the aspects and embodiments described above, theexpression-enhancing sequence can be placed in the indicated orientationas indicated in SEQ ID NO:1-6, or in the reverse of the orientationindicated in SEQ ID NO:1-6.

Any of the aspects and embodiments of the invention can be used inconjunction with any other aspect or embodiment of the invention, unlessotherwise specified or apparent from the context.

Other objects and advantages will become apparent from a review of theensuing detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Schematic diagram of a retroviral construct, pTE252, used forthe introduction of nucleic acid construct into a cell genome. LTR: longterminal repeat; LoxP: Cre recombinase recognition sequence:ATAACTTCGTATAATGTATGCTATACGAAGTTGT (SEQ ID NO:7); Lox511: a mutation ofLoxP sequences: ATAACTTCGTATAATGTATACTATACGAAGTTAG (SEQ ID NO:8); theLox511 sequence is recognized by Cre recombinase but the Lox511 sitedoes not recombine with a LoxP site. GFP: Green fluorescent protein; CMVMIE: human cytomegalovirus major immediate early promoter; npt: neomycinphosphotransferase; bla: beta lactamase; IRES: internal ribosomal entrysite.

FIG. 2 illustrates a plasmid construct used to identify an EESYR.

FIGS. 3A and 3B show an alignment of Chinese hamster ovary (CHO), mouse,human, and rat EESYR sequences for a fragment of SEQ ID NO:5.

FIG. 4 shows an alignment of SEQ ID NO:5 with mouse, human, and ratsequences.

FIG. 5 illustrates that an EESYR, operably linked to a gene of interest(GOI), exhibits enhanced expression over a GOI that is not operablylinked to an EESYR. For each plot, major ticks on the y-axis represent0, 300, 600, 900, and 1200; major ticks on the x-axis represent 10°,10¹, 10², 10³, and 10⁴.

FIG. 6 shows EESYRs compared in their relative ability to enhanceexpression of an operably linked GOI. For each plot, y-axis major ticksrepresent Counts of 0, 500, 1,000, 1,500, and 2,000; x-axis major ticksrepresent FITC of 10°, 101, 10², 10³, and 10⁴.

FIG. 7 illustrates clonal characteristics of cells with respect to EESYRfunctionality. For the top plot, major tick labels on both the y- andx-axes of the top plot represent 10°, 10¹, 10², 10³, and 10⁴; quadrantidentifiers are R10 and R11 (top, left to right) and R12 and R13(bottom, left to right). For the middle plot, which shows 96-well plateELISA results of sorted single cells, the y-axis of the center plotrepresents Titer (μg/ml), with major tick labels of 0, 2, 4, 6, 8, 10,12, and 14; whereas the x-axis represents Clone Number (in triplicate)from 1 to 63. For the bottom plot, which shows clone stability afterthree months without selection, y-axis major ticks represent incrementsof 100 from 0 to 1000. For the bottom plot, the left peak is for “Host,”whereas the right peak is for “Recombinant clones.”

FIG. 8 illustrates that EESYR cells undergo specific and efficientrecombination. In Panel B, y- and x-axes major ticks represent 10°, 10¹,10², 10³, and 10⁴; for each plot, quadrants are (left to right, top) R2and R3, and (left to right, bottom) R4 and R5.

FIG. 9 illustrates the rarity of random integration events in EESYRcells.

FIG. 10 illustrates testing of cis-acting elements employing EESYRsequences. Major ticks of the y- and x-axes major ticks represent 10°,10¹, 10², 10³, and 10⁴.

FIG. 11 illustrates testing promoters, introns, and UTRs using an EESYRsystem.

FIG. 12 illustrates optimizing protein expression using an EESYR system.

DETAILED DESCRIPTION

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, a reference to “a method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure.

Unless defined otherwise, or otherwise specified, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, particular methods and materials are now described. Allpublications mentioned herein are incorporated herein by reference intheir entirety.

DEFINITIONS

DNA regions are operably linked when they are functionally related toeach other. For example, a promoter is operably linked to a codingsequence if the promoter is capable of participating in thetranscription of the sequence; a ribosome-binding site is operablylinked to a coding sequence if it is positioned so as to permittranslation. Generally, operably linked can include, but does notrequire, contiguity. In the case of sequences such as secretory leaders,contiguity and proper placement in a reading frame are typical features.An EESYR is operably linked to a GOI where it is functionally related tothe gene of interest, for example, where its presence results inenhanced expression of the GOI.

The term “enhanced” when used to describe enhanced expression includesan enhancement of at least about 1.5-fold to at least about 2-foldenhancement in expression over what is typically observed by randomintegration into a genome, for example, as compared to a pool of randomintegrants of a single copy of the same expression construct.Fold-expression enhancement observed employing the sequences of theinvention is in comparison to an expression level of the same gene,measured under substantially the same conditions, in the absence of asequence of the invention. As used herein, the phrase“expression-enhancing” is used interchangeably with “enhanced expressionand stability” when referring to a region or sequence. An “enhancedexpression and stability region,” also referred to herein as an “EESYR,”is a region or sequence that exhibits more efficient recombination,insert stability, and higher level expression than is typically observedby random integration into a genome.

Enhanced recombination efficiency includes an enhancement of the abilityof a locus to recombine (for example, employing recombinase-recognitionsites). Enhancement refers to an efficiency over random recombination,which is typically 0.1%. A preferred enhanced recombination efficiencyis about 10-fold over random, or about 1%. Unless specified, the claimedinvention is not limited to a specific recombination efficiency.

Where the phrase “exogenously added gene” or “exogenously added GOI” isemployed with reference to a EESYR, the phrase refers to any gene notpresent within the EESYR as the EESYR is found in nature. For example,an “exogenously added gene” within a CHO EESYR (e.g., an EESYRcomprising a sequence of SEQ ID NO:5), can be a hamster gene not foundwithin the CHO EESYR in nature (i.e., a hamster gene from another locusin the hamster genome), a gene from any other species (e.g., a humangene), a chimeric gene (e.g., human/mouse), or any other gene not foundin nature to exist within the CHO EESYR.

Percent identity, when describing an EESYR, is meant to includehomologous sequences that display the recited identity along regions ofcontiguous homology, but the presence of gaps, deletions, or insertionsthat have no homolog in the compared sequence are not taken into accountin calculating percent identity. In explaining the usage of “percentidentity” in this context, the following sequence comparison (SEQ ID NO:9-SEQ ID NO: 12) will be referred to:

EESYR 5595 AGATTCTGTGGGCTCTGAGGCAACTTGACCTCAGCCAGATGGTATTTGAATAACCTGCTC5654 Rat Ch3 5619AGATTCTGTGGGTTCTGAGACAACTTGACTTCAGCCAGATGGCATTTGAATAAC------ 5672M Mus Ch2 6618AGATTCTGTGGGTTCTGAGACAACTTGACTTTAGCCAGATGGTATTTGAGTAATCTGGG- 6676H. Sap. Ch20 6620AGATTCAGTGGGCTTTGGGACAGCTTGACTTCAACTAGATGGTATTTGAATAATCTGCT- 6678****** ***** * ** * ** ****** * * * ****** ****** ***in which the “EESYR” 5595-5654 sequence is SEQ ID NO: 9, the “Rat Ch3”5619-5672 sequence is SEQ ID NO: 10, the “M Mus Ch2” 6618-6676 sequenceis SED ID NO: 11, and the “H. Sap. Ch20” sequence is SEQ ID NO: 12. Asused herein, a “percent identity” determination between the “EESYR”sequence above (for a CHO cell EESYR or fragment thereof) with a rathomolog (“Rat Ch3”) would not include a comparison of CHO sequences 5649through 5654, since the rat homolog has no homologous sequence tocompare in an alignment (i.e., the CHO EESYR has an insertion at thatpoint, or the rat homolog has a gap or deletion, as the case may be).Thus, in the comparison above, the percent identity comparison wouldextend from the “AGATTC” at the 5′ end to the “AATAAC” at the 3′ end. Inthat event, the rat homolog differs only in that it has a “T” at CHOEESYR position 5607, an “A” at CHO EESYR position 5614, a “T” at CHOEESYR position 524, and a “C” at CHO EESYR position 5637. Since thecomparison is over 54 contiguous bases in a 60 base pair stretch, withonly four differences (which are not gaps, deletions, or insertions),there is over 90% identity between the two sequences (CHO and rat) fromCHO EESYR position 5595 to CHO EESYR position 5654 (because “percentidentity” does not include penalties for gaps, deletions, andinsertions).

General Description

The invention is based at least in part on the discovery that there aresequences in a genome that exhibit more efficient recombination, insertstability, and higher level expression than other regions or sequencesin the genome. The invention is also based at least in part on thefinding that when such expression-enhancing sequences are identified, asuitable gene or construct can be exogenously added in or near thesequences and that the exogenously added gene can be advantageouslyexpressed. Such sequences, termed enhanced expression and stabilityregions (“EESYRs”), can be engineered to include recombinase recognitionsites for placement of genes of interest to create cell lines that arecapable of expressing proteins of interest. EESYRs can also be includedas in expression constructs such as, for example, expression vectors.Expression vectors comprising EESYRs can be used to express proteinstransiently, or can be integrated into a genome by random or targetedrecombination such as, for example, homologous recombination orrecombination mediated by recombinases that recognize specificrecombination sites (e.g., Cre-lox-mediated recombination). Expressionvectors comprising EESYRs can also be used to assess efficacy of otherDNA sequences, for example, cis-acting regulatory sequences.

The CHO EESYR described in detail herein was identified by randomintegration of DNA comprising lox sites into a CHO cell genome, followedby selection to identify sequences where expression was enhanced. Randomintegration and introduction of the lox site was achieved using aretroviral construct. Selection and screening were achieved using drugresistance markers and detectable labels (e.g., fluorescent proteinswith FACS screening), employing recombination methods that usedsite-specific recombination (e.g., lox sites and Cre recombinase).Selection continued until at least a 1.5- to 2-fold enhanced expressionover expression observed when randomly integrating an expressionconstruct into the CHO cell genome. Following identification of theEESYR, recombinase recognition sites (in the example provided, loxsites) were maintained in the EESYR for introducing expression cassettesthat comprise an expressible GOI, along with any other desirableelements such as, for example, promoters, enhancers, markers, operators,etc.

An illustration of a plasmid construct used in identifying an EESYRdisclosed in this application is shown in FIG. 2. The plasmid rescueconstruct comprises an expression cassette driven by an promoter,wherein the cassette is flanked on the 5′ and 3′ ends with recombinaserecognition sites (represented by ball-and-stick in FIG. 2). Insertionwithin an EESYR locus is shown, wherein the insertion results in theplasmid rescue construct replacing an expression cassette that comprisesa promoter and a marker, wherein the expression cassette within theEESYR locus is flanked on its 5′ and 3′ ends by recombinase recognitionsites (see FIG. 2).

Compositions and methods are provided for stably integrating a nucleicacid sequence into a eukaryotic cell, wherein the nucleic acid sequenceis capable of enhanced expression by virtue of being integrated in ornear an EESYR. Cells are provided that contain a recombinase recognitionsequence within or near an EESYR, convenient for inserting a GOI, inorder to achieve expression of a protein of interest from the GOI.Compositions and methods are also provided for using EESYRs inconnection with expression constructs, for example, expression vectors,and for adding an exogenous EESYR into a eukaryotic cell of interest.

Physical and Functional Characterization of an EESYR

The nucleic acid sequences referred to as EESYRs were empiricallyidentified by sequences upstream and downstream of the integration siteof a nucleic acid construct (comprising an expression cassette) of acell line expressing a reporter protein at a high level. The EESYRnucleic acid sequences of the invention provide sequences with a newfunctionality associated with enhanced expression of a nucleic acid (forexample, an exogenous nucleic acid comprising a GOI) that appear tofunction differently from that previously described for cis-actingelements such as promoters, enhancers, locus control regions, scaffoldattachment regions or matrix attachment regions. EESYRs do not appear tohave any open reading frames (ORFs), making it unlikely that EESYRsencode novel trans-activator proteins. Transfection experimentsdemonstrated that EESYR sequences display some characteristics ofcis-acting elements. EESYR activity is not detected in transienttransfection assays; EESYR sequences also appear to be distinct frompromoter and enhancer elements, which are detected with these methods.

Although EESYR sequences described in detail herein were isolated fromthe genome of two cell lines, EESYR sequences from these two cell linesare the same. EESYR activity was identified in a 6.472 kb fragment ofCHO genomic DNA 5′ with respect to a unique integration site of aretroviral vector comprising a DsRed reporter encoding sequence and in a7.045 kb fragment of CHO genomic DNA 3′ with respect to the integrationsite. Expression vectors comprising the isolated 6.472 kb region and theisolated 7.045 kb region and shorter fragments thereof were able toconfer upon CHO cells transfected with them high levels of expression ofrecombinant proteins.

The invention encompasses expression vectors comprising reverseorientated EESYR fragments. Reverse orientated EESYR fragments were alsocapable of conferring upon CHO cells transfected with them high levelsof expression of recombinant proteins.

Other combinations of the fragments described herein can also bedeveloped. Examples of other combinations of the fragments describedherein that can also be developed include sequences that includemultiple copies of the EESYR disclosed herein, or sequences derived bycombining the disclosed EESYR with other nucleotide sequences to achieveoptimal combinations of regulatory elements. Such combinations can becontiguously linked or arranged to provide optimal spacing of the EESYRfragments (e.g., by the introduction of spacer nucleotides between thefragments). Regulatory elements can also be arranged to provide optimalspacing of an EESYR with respect to the regulatory elements.

The EESYR sequences disclosed herein were isolated from CHO cells.Homologous expression-enhancing elements are expected to exist in cellsfrom other mammalian species (such as, for examples, humans; see FIG. 3)as well as in cell lines derived from other tissue types, and can beisolated by techniques that are well-known in the art, for example, bycross-species hybridization or PCR-based techniques. In addition,changes can be made in the nucleotide sequence set forth in SEQ IDNOs:1-6 by site-directed or random mutagenesis techniques that are wellknown in the art. The resulting EESYR variants can then be tested forEESYR activity as described herein. DNAs that are at least about 80%identical, preferably at least about 90% identical, more preferably atleast about 95% identical, most preferably least about 99% identical innucleotide sequence to SEQ ID NOs:1-6 or fragments thereof having EESYRactivity are isolatable by routine experimentation, and are expected toexhibit EESYR activity. For fragments of EESYR, percent identity refersto that portion of the reference native sequence that is found in theEESYR fragment. Accordingly, homologs of EESYR and variants of EESYR arealso encompassed by embodiments of the invention. FIGS. 3A and B show analignment of mouse, human, and rat sequences with varying homology to afragment of SEQ ID NO:5.

Cell populations expressing enhanced levels of a protein of interest canbe developed using the methods provided herein. The absolute level ofexpression will vary with the specific protein, depending on howefficiently the protein is processed by the cell. Cell pools developedwith EESYR are stable over time, and can be treated as stable cell linesfor most purposes. Cloning steps can be delayed until later in theprocess of development than is customary for recombinant proteins.

EESYRs and Expression-Enhancing Fragments Thereof

The EESYR genomic locus is conserved among human, mouse and rat genomes.FIG. 4 shows percent identity among EESYR sequences. EESYR sequences,homologous to the 13.515 kb of cloned CHO EESYR DNA of SEQ ID NO:5, wereidentified among the published human, rat and mouse genomes using BLAST.Sequences were aligned to determine the percent homology using MacVector(9.0). Twenty-five by increments of the alignment are graphed as thepercent identity among CHO, human, mouse and rat EESYR sequences foreach consecutive 25 bp segment. As shown in FIG. 4, the vertical linemarks the location of site-specific recombination events to expressrecombinant protein genes of interest. Percent identity of EESYRsequences adjacent to a site-specific recombination location in an EESYRis shown in panel B of FIG. 4. Ten base pair increments of the alignedsequences corresponding to nt 5022-6110 of a CHO cell EESYR sequence(nucleotides 5022 through 6110 of SEQ ID NO:5) are graphed as thepercent identity among CHO, human, mouse and rat EESYR sequences foreach consecutive 10 bp segment. Sequences were aligned using MacVector™9.0. As shown in panel B, a significant identity of sequence is presentin this fragment of the EESYR cloned from CHO cells. It should be notedthat the comparison of FIG. 4 indicates a length of about 1400 bases,whereas the sequence of SEQ ID NO:5 contains 13,515 bases. The FIG. 4bases appear to extend over a longer stretch due to the existence ofgaps. Nucleotide spans recited are those corresponding to numbering inSEQ ID NO:5 unless otherwise indicated. The span of nucleotides fromabout 6200 to about 7600 as shown in FIG. 4B corresponds to nucleotidesof SEQ ID NO:5 numbered about 5,200 to about 6,000.

Accordingly, the invention also includes an expression-enhancingfragment of a nucleotide sequence of SEQ ID NO:5, wherein theexpression-enhancing fragment includes the nucleotide sequence indicatedby positions about residues 5022 through about 6110 of SEQ ID NO:5, orabout 5218 through about 6048 of SEQ ID NO:5; or about 6200 throughabout 7600, about 6500 to about 7400, or about 6400 to about 6500 shownin panel B of FIG. 4. The invention also encompasses anexpression-enhancing fragment of a nucleotide sequence that is at least80% identical, preferably at least 90% identical, more preferably atleast 95% identical, most preferably at least 99% identical to thenucleotide sequence indicated by positions about 6200 through about7600, or about 6500 through about 7400, or about 6400 through about 6500shown in panel B of FIG. 4. The invention includes vectors comprisingsuch a fragment, including for transient or stable transfection. Theinvention also includes a eukaryotic cell comprising such a fragmentwherein the fragment is exogenous and is integrated into the cellgenome, and cells comprising such a fragment having at least onerecombinase recognition site that is within, immediately 5′, orimmediately 3′ to the fragment.

In one embodiment, the expression-enhancing fragment of SEQ ID NO:5 islocated at a position within SEQ ID NO:5 selected from nucleotidesspanning positions numbered 10-13,515; 20-12,020; 1,020-11,020;2,020-10,020; 3,020-9,020; 4,020-8,020; 5,020-7,020; 6,020-6,920;6,120-6,820; 6,220-6,720; 6,320-6,620; 6,420-6,520; 6,460-6,500;6,470-6,490; and 6,475-6,485.

In one embodiment, the EESYR is employed to enhance the expression of aGOI, as illustrated in FIG. 5. FIG. 5 shows a GOI operably linked with apromoter (with an upstream marker having its own promoter) integrated ina non-EESYR position in a CHO cell genome, and a FACS readout showingthe distribution of expression in a stably transfected population ofcells. In comparison, a GOI operably linked to a promoter integrated atan EESYR position in a CHO cell genome is shown, and a FACS readoutshowing the distribution of expression in a stably transfectedpopulation of cells is also shown. In this embodiment, the GOI expressedwithin the EESYR locus shows an enhanced expression of about two-fold incomparison to the GOI expressed at a non-EESYR locus.

In various embodiments, expression of a GOI can be enhanced by placingthe GOI within an EESYR, 5′ to an EESYR, or 3′ to an EESYR. The precisedistance between the GOI and the EESYR, where the GOI is either 5′ or 3′to the EESYR, should be such that the EESYR is operably linked to theGOI. An EESYR is operably linked to the GOI where expression of theGOI—at the selected distance from the EESYR (in the 5′ or 3′direction)—retains the ability to enhance expression of the GOI over,for example, expression typically observed due to a random integrationevent. In various embodiments, enhancement is at least about 1.5-fold toabout 2-fold or more. Preferably, enhancement in expression as comparedto a random integration, or random expression, is about 2-fold or more.

FIG. 6 shows an embodiment wherein SEQ ID NO:1 (“EESYR 5”) and SEQ IDNO:2 (“EESYR 3′”) are compared in their relative ability to enhanceexpression of an operably linked GOI, wherein the GOI is operably linkedto a promoter as well (a marker and a promoter operably linked to themarker are shown 5′ to the GOI promoter). Orientation of the EESYR isshown by the direction of the arrow beneath the term “EESYR.” Theconstructs are randomly integrated into a CHO cell genome. Expression isrelative to the randomly integrated construct that does not comprise anyEESYR. FACS readouts showing relative expression are shown on the right.In FIG. 6, the first EESYR construct employs SEQ ID NO:1; the secondEESYR construct employs SEQ ID NO:3; the third EESYR construct is SEQ IDNO:2; the fourth EESYR construct is SEQ ID NO:4. As shown in the figure,SEQ ID NO:3 displays a 2.4-fold enhancement in expression, and SEQ IDNO:1 displays a 2-fold enhancement of expression.

EESYR recombinant cell pools display clonal characteristics. FIG. 7illustrates clonal characteristics of EESYR recombinant pools. In thetwo-color FACS plot representing a dual parameter histogram of cellslabeled with red or green markers (red cells are host CHO cells; greencells are recombinants expressing a GOI), EESYR recombinant pools showclustering in the plot that reflects substantially identical growth andexpression, flow cytometry profile, and Southern analysis (not shown). Ahistogram of ELISAs of single cells demonstrate uniform expression inall clones. Clonal stability is also high after three months withoutselection.

EESYR recombinants undergo specific and efficient recombination, asshown in FIG. 8. Panel A shows two markers separated by an IRES andflanked by recombinase recognition sites, and a third marker not flankedby recombinase recognition sites as a random integration control. Whenrecombined at an EESYR locus comprising a marker flanked by tworecombinase recognition sites, recombination is specific. Panel B showslittle random integration in the absence of recombinase, but efficientand site-specific integration in the presence of recombinase.

Random integration using site specific recombination at an EESYR is rare(see FIG. 9). FIG. 9 shows that when random integration events arevisualized, such events represent only a tiny fraction of integrationevents.

In another embodiment, EESYR cis-acting elements can be assessed usingthe methods and compositions of the invention. As shown in FIG. 10,EESYR recombinant cells, allows comparison of cis-acting elementsequivalently. Because EESYR recombinants behave as a clonal population,differences in gene expression as the result of, for example, thepresence or absence of suspected cis-acting elements, can be directlycompared. Isogenic cell lines allow direct comparison of cis-actingelements. Using an EESYR system, cis-acting elements, such as, forexample, promoters, introns, and UTRs, are preferably located betweenrecombination sites. Expression optimization can also be achieved,including, for example, expression cassette orientation and codonoptimization. By way of example, FIG. 11 shows cassettes flanked withrecombination recognition sites that contain a promoter, a marker,various cis-elements (here, introns in panel A; UTRs in panel B), and aGOI were integrated at an EESYR (SEQ ID NO: 1 at the 5′ end, SEQ ID NO:2at the 3′ end). Relative expression of the GOI is shown on the right.

FIG. 12 shows an example of how protein optimization can be achievedusing the methods and compositions of the invention. FIG. 12 confirmsthat optional placement of a cDNA for a light chain antibody gene is 5′to the cDNA for a heavy chain antibody gene.

Proteins of Interest

A nucleic acid sequence encoding a protein of interest can beconveniently integrated into a cell comprising an EESYR having arecombinase recognition site through, for example, arecombinase-mediated cassette exchange (RMCE) process. Any protein ofinterest suitable for expression in eukaryotic cells can be used. Forexample, the protein of interest can be an antibody or fragment thereof,a chimeric antibody or fragment thereof, an ScFv or fragment thereof, anFc-tagged protein or fragment thereof, a growth factor or a fragmentthereof, a cytokine or a fragment thereof, or an extracellular domain ofa cell surface receptor or fragment thereof.

Nucleic Acid Constructs

Recombinant expression vectors can comprise synthetic or cDNA-derivedDNA fragments encoding a protein, operably linked to a suitabletranscriptional and/or translational regulatory element derived frommammalian, viral or insect genes. Such regulatory elements includetranscriptional promoters, enhancers, sequences encoding suitable mRNAribosomal binding sites, and sequences that control the termination oftranscription and translation, as described in detail below. Mammalianexpression vectors can also comprise nontranscribed elements such as anorigin of replication, other 5′ or 3′ flanking nontranscribed sequences,and 5′ or 3′ nontranslated sequences such as splice donor and acceptorsites. A selectable marker gene to facilitate recognition oftransfectants may also be incorporated.

Transcriptional and translational control sequences in expressionvectors useful for transfecting vertebrate cells may be provided byviral sources. For example, commonly used promoters and enhancers arederived from viruses such as polyoma, adenovirus 2, simian virus 40(SV40), and human cytomegalovirus (CMV). Viral genomic promoters,control and/or signal sequences may be utilized to drive expression,provided such control sequences are compatible with the host cellchosen. Non-viral cellular promoters can also be used (e.g., theβ-globin and the EF-1α promoters), depending on the cell type in whichthe recombinant protein is to be expressed.

DNA sequences derived from the SV40 viral genome, for example, the SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites may be used to provide other genetic elements useful forexpression of a heterologous DNA sequence. Early and late promoters areparticularly useful because both are obtained easily from the SV40 virusas a fragment that also comprises the SV40 viral origin of replication(Fiers et al., Nature 273:113, 1978). Smaller or larger SV40 fragmentsmay also be used. Typically, the approximately 250 bp sequence extendingfrom the Hind III site toward the BglI site located in the SV40 originof replication is included.

Bicistronic expression vectors used for the expression of multipletranscripts have been described previously (Kim S. K. and Wold B. J.,Cell 42:129, 1985; Kaufman et al. 1991, supra) and can be used incombination with an EESYR sequence of the invention. Other types ofexpression vectors will also be useful, for example, those described inU.S. Pat. No. 4,634,665 (Axel et al.) and U.S. Pat. No. 4,656,134(Ringold et al.).

Host Cells and Transfection

The eukaryotic host cells used in the methods of the invention aremammalian host cells, including, for example, CHO cells and mouse cells.In a preferred embodiment, the invention provides a nucleic acidsequence that encodes an EESYR sequence from CHO cell. An integrationsite, for example, a recombinase recognition site, can be placed withinan EESYR, or 5′ or 3′ to the EESYR sequence. One example of a suitableintegration site is a lox p site. Another example of a suitableintegration site is two recombinase recognition sites, for example, alox p site and a lox 5511 site. In one embodiment, the EESYR sequence islocated on chromosome 6 of a CHO cell genome. In specific embodiments,the EESYR sequence is located within a sequence selected from the groupconsisting of nucleic acids comprising nucleotides 1-6473 and 4607-6473of SEQ ID NO: 1; and 1-7045, 1-3115, 1-2245, 1-935, and 1-465 of SEQ IDNO:2.

The invention includes a mammalian host cell transfected with anexpression vector of the invention. While any mammalian cell may beused, the host cell is preferably a CHO cell.

Transfected host cells include cells that have been transfected withexpression vectors that comprise a sequence encoding a protein orpolypeptide. Expressed proteins will preferably be secreted into theculture medium, depending on the nucleic acid sequence selected, but maybe retained in the cell or deposited in the cell membrane. Variousmammalian cell culture systems can be employed to express recombinantproteins. Examples of suitable mammalian host cell lines include theCOS-7 lines of monkey kidney cells, described by Gluzman (1981) Cell23:175, and other cell lines capable of expressing an appropriate vectorincluding, for example, CV-1/EBNA (ATCC CRL 10478), L cells, C127, 3T3,CHO, HeLa and BHK cell lines. Other cell lines developed for specificselection or amplification schemes will also be useful with the methodsand compositions provided herein. A preferred cell line is the CHO cellline designated K1. In order to achieve the goal of high volumeproduction of recombinant proteins, the host cell line should bepre-adapted to bioreactor medium in the appropriate case.

Several transfection protocols are known in the art, and are reviewed inKaufman (1988) Meth. Enzymology 185:537. The transfection protocolchosen will depend on the host cell type and the nature of the GOI, andcan be chosen based upon routine experimentation. The basic requirementsof any such protocol are first to introduce DNA encoding the protein ofinterest into a suitable host cell, and then to identify and isolatehost cells which have incorporated the heterologous DNA in a relativelystable, expressible manner.

One commonly used method of introducing heterologous DNA into a cell iscalcium phosphate precipitation, for example, as described by Wigler etal. (Proc. Natl. Acad. Sci. USA 77:3567, 1980). DNA introduced into ahost cell by this method frequently undergoes rearrangement, making thisprocedure useful for cotransfection of independent genes.

Polyethylene-induced fusion of bacterial protoplasts with mammaliancells (Schaffner et al., (1980) Proc. Natl. Acad. Sci. USA 77:2163) isanother useful method of introducing heterologous DNA. Protoplast fusionprotocols frequently yield multiple copies of the plasmid DNA integratedinto the mammalian host cell genome, and this technique requires theselection and amplification marker to be on the same plasmid as the GOI.

Electroporation can also be used to introduce DNA directly into thecytoplasm of a host cell, for example, as described by Potter et al.(Proc. Natl. Acad. Sci. USA 81:7161, 1988) or Shigekawa et al.(BioTechniques 6:742, 1988). Unlike protoplast fusion, electroporationdoes not require the selection marker and the GOI to be on the sameplasmid.

More recently, several reagents useful for introducing heterologous DNAinto a mammalian cell have been described. These include Lipofectin™Reagent and Lipofectamine™ Reagent (Gibco BRL, Gaithersburg, Md.). Bothof these reagents are commercially available reagents used to formlipid-nucleic acid complexes (or liposomes) which, when applied tocultured cells, facilitate uptake of the nucleic acid into the cells.

A method for amplifying the GOI is also desirable for expression of therecombinant protein, and typically involves the use of a selectionmarker (reviewed in Kaufman supra). Resistance to cytotoxic drugs is thecharacteristic most frequently used as a selection marker, and can bethe result of either a dominant trait (e.g., can be used independent ofhost cell type) or a recessive trait (e.g., useful in particular hostcell types that are deficient in whatever activity is being selectedfor). Several amplifiable markers are suitable for use in the expressionvectors of the invention (e.g., as described in Maniatis, MolecularBiology: A Laboratory Manual, Cold Spring Harbor Laboratory, N Y, 1989;pgs 16.9-16.14).

Useful selectable markers for gene amplification in drug-resistantmammalian cells are shown in Table 1 of Kaufman, R. J., supra, andinclude DHFR-MTX resistance, P-glycoprotein and multiple drug resistance(MDR)-various lipophilic cytotoxic agents (e.g., adriamycin, colchicine,vincristine), and adenosine deaminase (ADA)-Xyl-A or adenosine and2′-deoxycoformycin.

Other dominant selectable markers include microbially derived antibioticresistance genes, for example neomycin, kanamycin or hygromycinresistance. However, these selection markers have not been shown to beamplifiable (Kaufman, R. J., supra). Several suitable selection systemsexist for mammalian hosts (Maniatis supra, pgs 16.9-16.15).Co-transfection protocols employing two dominant selectable markers havealso been described (Okayama and Berg, Mol. Cell Biol 5:1136, 1985).

Useful regulatory elements, described previously or known in the art,can also be included in the nucleic acid constructs used to transfectmammalian cells. The transfection protocol chosen and the elementsselected for use therein will depend on the type of host cell used.Those of skill in the art are aware of numerous different protocols andhost cells, and can select an appropriate system for expression of adesired protein, based on the requirements of the cell culture systemused.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention and are not intended to be limitingthereof.

Examples

The following examples are put forth so as to provide those of ordinaryskill in the art how to make and use the methods and compositionsdescribed herein, and are not intended to limit the scope of what theinventors regard as their invention. Efforts have been made to ensureaccuracy with respect to numbers used (e.g., amount, temperature, etc.)but some experimental error and deviation should be accounted for.Unless indicated otherwise, parts are parts by weight, molecular weightis average molecular weight, temperature is in degrees Centigrade, andpressure is at or near atmospheric.

Example 1 Generation of RGC9 and RGC16 Cell Lines

CHO K1 cells (1×10⁷) were infected with pantropic retrovirus producedwith plasmid pTE252 (FIG. 1), having a lox p site in it, at an MOI ofless than 1 to generate a stable pool of cells with mostly oneretroviral insertion per cell. Cells in the stable pool that expressed amarker protein at a high level were selected and expanded. Selectionrounds were conducted to identify cell populations capable of enhancedexpression. Thirty-six clones were isolated and expanded into 36 celllines. Clones exhibiting the highest recombination efficiency wereidentified and cloned, wherein the clones each contained at least onerecombinase recognition site in an enhanced expression locus. Eight cellpopulations with the best recombination efficiency were selected, andreassessed for enhanced protein expression. Two cell populations wereselected and designated RGC9 and RGC16. Southern blot analysis of thecell populations from the original 36 cell lines that corresponded tothese two cell lines showed that a single copy of a reporter constructwas integrated into the CHO cell genome at the same locus in the case ofboth cell populations, and the location of integration was determined tobe at the triplet “act” at nucleotide position 6,471 to 6,473 of SEQ IDNO:5. At least one of these two cell lines were employed in experimentsdescribed below.

Example 2 Expression of FcFP1 Protein in Serum-Free Production Medium

RGC38 cells were derived from RGC9 cells and were adapted to grow insuspension in a serum-free production medium. RGC38 cells were used ashost cells for the expression of FcFP1 protein (Fc fusion protein-1).RGC38 cells were transfected in a ten-centimeter plate with a FcFP1expression vector, pTE851 and a Cre plasmid, pRG858. The FcFP1 plasmidhas, in 5′ to 3′ direction, a LoxP site, a SV40 late promoter, ahygromycin resistant gene, an IRES, an eGFP, a CMV MIE promoter, a geneencoding a FcFP1 protein, and a Lox511 site. Cells were cultured in F12medium with 400 μg/ml hygromycin for two weeks after transfection. Cellsexpressing eGFP but not DsRed were isolated using flow cytometry anddesignated as RS421-1. Isolated cells were essentially isogenic, thoughderived from different founder cells. RS421-1 cells were expanded insuspension cultures in serum-free production medium. FcP1 protein inconditioned medium of 3-day old cultures was examined in SDS-PAGE withCoomassie blue staining. FcF1 protein in the conditioned medium wasabundant and could be seen without purification.

Example 3 Regulated Expression of FcFP1 Protein in Serum-Free ProductionMedium

RGC49 cells were derived from RGC16, were adapted to grow in serum-freeproduction medium, and contained a stably integrated tetR-YFP expressionplasmid, pcDNA6/TR. The tetR protein allows regulation of transcriptionfrom promoters that comprise a tet operator sequence by tetracycline ordoxycycline. RGC49 cells were co-transfected with pTE851 and pRG858. Thetransfected cells were selected with 400 μg/ml hygromycin for two weeks.Cells expressing eGFP but not YFP were isolated using flow cytometry anddesignated as RS569-1. RS569-1 cells were expanded in suspensioncultures in serum-free production medium in the presence or absence ofdoxycycline. FcP1 protein in conditioned medium of 3-day old cultureswas examined by SDS-PAGE and Coomassie blue staining. The RS569-1 cellsexpressed FcFP1 protein similarly to RS421-1 upon in the presence of 1μg/ml doxycycline in the culture medium. Very little FcFP1 protein wasdetected from the RS569-1 cells in the absence of doxycycline.

Example 4 Dual Lox Cell Line Construction

RGC23 cells were derived from RGC16, were adapted to grow in Sigma CHOSSM serum-free medium (Saint Louis, Mo.), and carried DsRed. RGC23 cellswere transfected with a Cre plasmid, pRG858 and a eGFP and FcFP2 proteinexpression vector, pTE357. The FcFP2 vector has, in 5′ to 3′ direction,a LoxP site, a SV40 late promoter, a hygromycin resistant gene, a CMVMIE promoter, a gene encoding FcFP2 protein, an IRES, an eGFP, and aLox511 site. Cells expressing both DsRed and eGFP were collected and asingle cell was isolated. The isolated cell was expanded in culture, andthe resulting cell line was designated RS398-2-6. RS398-2-6 was thentransfected with pRG858 (Cre plasmid) and pRG1231, a eCFP expressionplasmid. pRG1231 has, in 5′ to 3′ direction, a LoxP site, a CMV MIEpromoter, a puromycin resistant gene, an IRES, an eCFP, and a Lox511site. Cells expressing DsRed and eCFP but not eGFP were isolated by flowcytometry as a pool and designated as RS630.

Example 5 Antibody Heavy Chain and Light Chain Expression Using a DualLox Cell Line

RS630 cells were transfected with pTE828, a 15G1 antibody heavy chainand eGFP expression vector, pTE829, a 15G1 antibody light chain and eYFPexpression vector, and pRG858. pTE828 has, in 5′ to 3′ direction, a LoxPsite, a SV40 late promoter, a hygromycin resistant gene, an IRES, aneGFP, a CMV MIE promoter, the heavy chain gene of 15G1 antibody, and aLox511 site. pTE829 has, in 5′ to 3′ direction, a LoxP site, a SV40 latepromoter, a neomycin resistant gene, an IRES, an eYFP, a CMV MIEpromoter, the light chain gene of 15G1 antibody, and a Lox511 site. Thetransfected cultures were selected with hygromycin and G418 at 400 μg/mleach for two weeks. Cells expressed both YFP and eGFP but neither dsRednor eCFP were isolated by flow cytometry. The isolated cells wereexpanded in suspension culture in serum-free production medium and weredesignated as RS631 cells. Aliquots of conditioned medium from 3-day oldculture were analyzed by SDS-PAGE. The antibody products from RS631cells were readily detected by Coomassie blue staining.

Example 6 Use of a Third Lox Site (Lox2272) at EESYR to Create DualExpression Cassette

Cells from any cell line carrying a DsRED gene flanked by a LoxP siteand a Lox511 site at EESYR locus are transfected with pRG858 and avector comprising, in 5′ to 3′ direction, a LoxP site, a first promoter,a YFP, a Lox2272 site, a second promoter, an eGFP, and a Lox511. Cellsexpressing eGFP and YFP, but not DsRed are isolated. Isolated cells arethen transfected with pRG858, pRG1167 (a vector that has, in 5′ to 3′direction, a LoxP site, a SV40 late promoter, a hygromycin resistantgene, a CMV MIE promoter, a DsRed and a Lox2272 site) or pRG1234 (avector that has, in 5′ to 3′ direction, a Lox2272 site, a SV40 latepromoter, a hygromycin resistant gene, a CMV MIE promoter, a DsRed and aLox511 site). Cells capable of expressing either DsRED and eGFP but notYFP, or DsRED and YFP but not eGFP, are isolated.

Example 7 Antibody Expression from RGC38 Host Cells

RGC38 cells were transfected with pTE963 and pRG858. pTE963 has, in 5′to 3′ direction, a LoxP site, a SV40 late promoter, a hygromycinresistant gene, an IRES, an eGFP, a CMV MIE promoter, the light chaingene of 15G1 antibody, a CMV MIE promoter, the heavy chain gene of 15G1antibody, and a Lox511 site. The transfected cultures were selected withhygromycin at 400 μg/ml each for two weeks. Cells that expressed eGFPbut not dsRed were isolated by flow cytometry. The isolated cells wereexpanded in suspension in serum-free production medium and were named asRS533 cells. For the production of 15G1 antibodies, RS533 cells werecultured in a bioreactor for 10 days. Aliquots of spent medium from daysix to day ten were collected and their protein composition was analyzedby SDS-PAGE. The heavy chain and light chain peptide of the 15G1antibody in the reactor spent medium were readily detected by Coomassieblue staining.

Example 8 Rescuing and Subcloning EESYR Sequences

A CHO cell line (designated RGC21) expressing high levels of a reportergene, DsRed, was selected for isolation of EESYR sequences, sinceSouthern blot analysis indicated that the high expression of DsRedexpression observed for this cell line is driven by a single integrationof an expression cassette encoding DsRed. Genomic sequences 5′ to theexpression cassette were rescued by transfecting RGC21 cells withlinearized pTE494 plasmids, a vector that has, in 5′ to 3′ direction, aLoxP site, an ampicillin resistance gene, a bacterial origin orreplication, a CMV MIE promoter, a neomycin phosphotransferase gene, anIRES, an eGFP and a Lox511 site. Cells expressing eGFP but not DsRedwere isolated by flow cytometry as a pool. Genomic DNA was isolated,digested with XbaI restriction endonuclease, and self ligated to createpRG1106. Genomic sequences 3′ to the expression cassette were rescued bytransfecting RGC21 cells with circular pTE495 plasmids, a vector thathas, in 5′ to 3′ direction, a LoxP site, a CMV MIE promoter, a neomycinphosphotransferase gene, an IRES, an eGFP, a bacterial origin orreplication, an ampicillin resistance gene, and a Lox511 site. Cellsexpressing eGFP but not DsRed were isolated by flow cytometry as a pool.Genomic DNA was isolated, digested with MfeI restriction endonuclease,and self ligated to create pRG1099.

Example 9 Plasmid Construction for EESYR Analysis

EESYR sequences were excised from either pRG1106 or pRG1099 as AgeIfragments and inserted into the AgeI and NgoMIV sites of pTE575, aplasmid expressing FCFP2, to yield plasmid pTE809. The pTE575 plasmidhas, in 5′ to 3′ direction, a SV40 late promoter, a hygromycin resistantgene, a CMV MIE promoter, and a gene encoding FCFP2 protein. In stablytransfected CHO cells, pTE809 and pTE575 yielded 97.22% and 38.57% ofcells expressing detectable levels of FCFP2 protein, respectively. Themean fluorescence of FcFP2 detected by an FITC conjugated antibody was482.54 and 279.75 for cultures transfected with pTE809 and pTE575,respectively. Thus, the inclusion of EESYR in expression vectorsincreased the expression of FCFP2 protein in stable transfection.

The present invention may be embodied in other specific embodimentswithout departing from the spirit or essence thereof.

We claim:
 1. A method of making a recombinant cell comprisingintroducing at least one exogenous sequence into an expression-enhancingexpression and stability region (EESYR) sequence, or 5′ or 3′ of anEESYR sequence, wherein the EESYR sequence is selected from the groupconsisting of SEQ ID NO:1-6.
 2. The method of claim 1, wherein the atleast one exogenous sequence comprises at least one of an integrationsite and a gene of interest (GOI).
 3. The method of claim 2, wherein theintegration site is a recombinase recognition site.
 4. The method ofclaim 2, wherein the integration site facilitates recombination.
 5. Themethod of claim 1, wherein the exogenous sequence is introduced into theEESYR sequence, or 5′ or 3′ of an EESYR sequence, by homologousrecombination, site-directed integration, random mutagenesis orintegrase technology.
 6. The method of claim 3, wherein the recombinaserecognition site is selected from the group consisting of a LoxP site, aLox511 site, a Lox2272, and a Frt site.
 7. The method of claim 2,wherein a gene of interest (GOI) is immediately adjacent to anintegration site and operably linked to the expression-enhancingsequence.
 8. The method of claim 2, wherein the GOI encodes a proteinselected from an immunoglobulin or an antigen-binding fragment thereof,an Fc fusion protein, and a receptor or ligand-binding fragment thereof.9. The method of claim 8, wherein the immunoglobulin is selected from anantibody light chain or antigen-binding fragment thereof, an antibodyheavy chain or antigen-binding fragment thereof, or an Fc fusionprotein.
 10. The method of claim 8, wherein expression of the GOIintroduced into an EESYR sequence is enhanced at least 2-fold comparedto the same GOI that is not operably linked to an EESYR sequence. 11.The method of claim 7, wherein the GOI is immediately adjacent and 5′ ofa recombinase recognition site, and further comprising a secondrecombinase recognition site immediately adjacent and 3′ of the GOI. 12.The method of claim 11, further comprising a second GOI immediatelyadjacent and 3′ of the second recombinase recognition site.
 13. Themethod of claim 12, further comprising a third recombinase recognitionsite immediately adjacent and 3′ of the second GOI.
 14. The method ofclaim 2, further comprising at least one marker gene.
 15. The method ofclaim 14, wherein the at least one marker gene is selected from thegroup consisting of a drug resistance gene and an expression reportergene.
 16. The method of claim 2, wherein the GOI is operably linked to apromoter.
 17. The method of claim 12, further comprising a promoteroperably linked to the first GOI and a promoter operably linked to thesecond GOI.
 18. The method of claim 17, wherein the first GOI encodes alight chain of an antibody and the second GOI encodes a heavy chain ofan antibody.
 19. The method of claim 17, wherein the first GOI encodes aheavy chain of an antibody and the second GOI encodes a light chain ofan antibody.
 20. The method of claim 13, wherein the second and thethird recombinase recognition sites are in an orientation opposite tothe first recombinase recognition site.
 21. The method of claim 13,wherein the first, second, and third recombinase recognition sites aredifferent.
 21. The method of claim 16, wherein the promoter is operablylinked to an operator.
 22. The method of claim 22, wherein the promotercomprises a eukaryotic promoter, and the eukaryotic promoter is operablylinked to a prokaryotic operator.
 23. A vector for use in making arecombinant cell, wherein the vector comprises a sequence homologous toan expression-enhancing expression and stability region (EESYR) sequenceselected from the group consisting of SEQ ID NO:1-6, or a fragmentthereof, and at least one exogenously added gene.
 25. The vector ofclaim 24, wherein the EESYR sequence is selected from the groupconsisting of SEQ ID NO:1, 2, 3, 4, and a combination thereof.
 26. Thevector of claim 24, wherein the vector comprises a 5′ homology arm and a3′ homology arm, wherein each homology arm is homologous to a sequencewithin any one of SEQ ID NO:1-6.
 27. The vector of claim 26, wherein the5′ and 3′ homology arms are homologous to sequences within SEQ ID NO:5.28. The vector of claim 24, wherein the vector comprises one, two,three, four, or five or more exogenously added genes.
 29. The vector ofclaim 28, wherein the one or more exogenous genes are selected from thegroup consisting of a transcriptional regulatory gene, translationalregulatory gene, promoter, enhancer, ribosomal binding site, selectablemarker gene and gene of interest (GOI).
 30. The vector of claim 29,wherein, the exogenous GOI is operably linked to an exogenous promoter.31. The vector of claim 29, wherein each of the two, three, four, orfive or more exogenous genes are each operably linked to a promoter. 32.The vector of claim 29, wherein the cell is a CHO cell.
 33. The vectorof claim 29, wherein the cell is a CHO cell, the at least one exogenousGOI is a human gene, and the human gene is operably linked to anexogenous promoter.
 34. The vector of claim 29, wherein the first GOIencodes a light chain of an antibody and the second GOI encodes a heavychain of an antibody.